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Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful method for molecule-specific imaging which uses molecular vibrations as contrast mechanism [1]. The specimen under investigation is illuminated by two light fields, called pump and Stokes signal. Their frequency difference, i.e. the so-called Stokes-shift, is tuned to a Raman-active transition of the chemical compound of interest (Fig. 1). Now, the pump signal is inelastically scattered at the generated collective material excitation. This results in the emission of a so-called anti-Stokes (AS) signal which is blue shifted with respect to the pump signal.
Fig. 1: Energy scheme for coherent anti-Stokes Raman scattering. The Stokes-shift is tuned to a Raman-active transition of the molecules under investigation. The dashed lines represent virtual energy levels.

For conventional CARS microscopy the AS signal is directly detected by a highly sensitive photodetector while the sample is raster scanned relative to the fixed excitation beams. CARS microscopy has various applications, e.g. examination of unstained live cells or chemical imaging of exposed photoresist on semiconductor surfaced with high spatial resolution.

CARS microscopy is a priori not background free which results in contrast reduction due to nonresonant background signals. In addition, for aqueous solutions strong resonant background signals may be present due to the broad Raman band of water. Therefore, weak AS signals from small scatterers are often overwhelmed by these background signals.

We use a novel time-resolved heterodyne detection scheme for CARS microscopy, referred to as ‘gated heterodyne CARS’ (GH-CARS), which is capable of providing a significantly higher vibrational contrast than conventional CARS microscopy. Furthermore such a detection scheme allows very narrow-band filtering and offers heterodyne gain. Moreover, shot-noise-limited detection can be achieved even in the presence of strong incoherent background signals, e.g. during combustion processes.

In order to accomplish GH-CARS, the AS signal is superimposed with a strong field of the same frequency, the so-called local oscillator (LO). Both fields interfere and the resulting interference amplitude is extracted. Because CARS is a coherent four-wave-mixing process, such a heterodyne detection becomes practicable as soon as an LO with a fixed phase relationship to pump and Stokes fields is available.

In cooperation with the group of Prof. Dr. Eberhard Riedle, LMU München, we use three noncollinearly phase-matched optical parametric amplifiers (NOPAs) [2] as phase-coherent light source for pump, Stokes and LO field. The NOPAs are seeded by a common white-light continuum generated by focusing ultra-short light pulses (150 fs) into a sapphire plate.

Fig. 2 shows a typical spatial interference pattern resulting from the superposition of AS and LO beam. The temporally stable high-contrast interferogram proves the phase coherence of the white-light which is preserved by the parametric amplification.

Fig. 2: Superposition of anti-Stokes field and local oscillator. The anti-Stokes signal comes from the excitation of the symmetric C-D stretching vibration of deuterated benzene (C₆D₆)

The use of a pulsed light source for the CARS process and the LO allows a time-resolved detection. To this end, an additional light pulse which is time-delayed with respect to the excitation pulses is focussed into the sample. As long as the collective excitation has not completely decayed yet, a second inelastic scattering process occurs resulting in the emission of a second AS signal. An appropriately delayed LO pulse facilitates the separation of the second from the first AS pulse. Thus, if the collective material excitation of the sample dephases much slower than that one of the solvent, the technique enables an efficient suppression of background signals. For deuterated benzene (sample) and heavy water (solvent) the signal-to-background ratio can be enhanced by a factor of >100 [3].
The gating mechanism described above is also applicable for the suppression of true nonresonant background signals due to the associated rapid dephasing.

The GH-CARS concept can be applied to carry out high-contrast CARS microscopy. Fig. 3 shows GH-CARS images of 10-µm polystyrene beads embedded in water. In order to excite the aromatic CH vibration of the polystyrene the Stokes-shift is tuned to 3052 cm^-1. Fig. 3 (a) displays the GH-CARS images in the case of a LO pulse which is not delayed. A large background signal from the water molecules is found from the intensity profile across the beads. Contrary to that, the intensity profile shows a much higher signal-to-background ratio if the LO pulse is delayed (Fig. 3 (b)). 

Fig. 3: GH-CARS images of 10-µm polystyrene beads embedded in water. (a) LO pulse not delayed, (b) LO pulse delayed for 530 fs. Each image consists of 100x35 (pixel)². The intensity profiles across the lines indicated by the arrows are shown below the images. The signal reduction at the edge of the beads can be explained by reflection and scattering losses.

A possible application of GH-CARS microscopy could be high-contrast imaging of microscopic benzene droplets in ground water. Because of the high chemical selectivity of CARS one could even distinguish between different derivatives of the benzene. Such an investigation would be unrealisable with conventional light-optical and CARS microscopes.

We work on the project “Heterodyne CARS Microscopy” in collaboration with working group 4.23 (Ultra-High Resolution Microscopy).

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© Physikalisch-Technische Bundesanstalt Page created: 2004-02-05, last update: 2006-05-22, Yvonne Zimber